The present invention provides a method of retarding the differentiation of a biological cell. The biological cell may preferably comprise a stem or progenitor cell. The invention further provides cell culture media that may be used to retard differentiation of cultured cells, biological cells comprising constructs useful in the retardation of cell differentiation, and methods for the therapeutic manipulation of biological cells.
The production, maintenance and use of stem and progenitor cells is currently the subject of much scientific interest and research. Stem and progenitor cells constitute a highly valuable system for studying aspects of development and have the potential to revolutionise the treatment of injury and disease as the basis of cellular therapies.
The therapeutic use of stem cells offers a powerful new therapeutic approach as compared to existing drug-based therapies. This new approach may have applications in degenerative illnesses (e.g. Alzheimer), cardiovascular diseases, cancer and diseases of the nervous system (e.g. Multiple sclerosis). Many such diseases are currently untreatable.
Disease management is currently achieved through the use of disease modifying drugs, often whose activities the body often poorly tolerates. In addition, these drugs are limited in their ability to control only the symptoms of a disease and are unable to offer cures. Poor disease coverage coupled with the failings in current drug based therapies is driving the quest for new disease management methods and treatments.
The most promising development towards such goals is in the development of regenerative tissue engineering stem cell based therapies. In fact, the potential of stem cells as therapeutic cures is well known; with over 10,000 individuals undergoing successful bone marrow stem cell transplantations yearly in the UK.
Estimations currently predict an explosion in the stem cell market, reaching a value in the region of $10 billion by 2010.
The therapeutic use of stem cells relies on the ability of these cells to give rise to multiple tissue types. Accordingly such cells are able to generate replacement tissue in subjects to which they are administered.
Stem cells such as human embryonic stem (hES) cells are widely believed to have the capability to revolutionize disease therapeutics with the potential of meeting many of the unmet medical needs. Embryonic stem cells are unique in their ability to develop and differentiate into all the cells and tissues of the body. As such, they are a potential source of replacement cells and tissues for organ repair in chronic diseases. The unique characteristics that commend embryonic stem cells to therapeutic use are:                i) They are unspecialised cells capable of proliferation and self-renewal.        ii) Under specific physiological conditions they can be induced to become cells with specific functions, such as beating cells of the heart.        
Current protocols for the culture and growth of stem cells (such as from stem cells from mouse and human sources) requires the involvement of skilled technicians, as well as the use of specialized cell culture media intended to maximise the yield of pluripotent or multipotent cells within such cultures.
However, current techniques for the culture of stem or progenitor cells are subject to spontaneous differentiation of the cultured cells which gives rise to the development of various differentiated cell types. Such spontaneous differentiation severely decreases the yield of pluripotent or multipotent cells over extended passages, a decrease that is particularly notable in human cell cultures. If cells to be used for therapy undergo uncontrolled differentiation during culture the number of possible lineages into which they may develop, and hence their ultimate therapeutic potential, is reduced. Therefore, for ES cells to realise their potential in cellular therapy applications it is essential that increased yields of pluripotent cells are achievable using cost-effective medium, absence of animal products (such as serum) and minimum technical requirements.
Stem cells offer the promise of treatment, and possibly, the cure of a broad array of human diseases, benefiting patients, family members, physicians and society in general.
Current approaches to maintaining the undifferentiated phenotype of ES cells are focused on the identification of exogenous and endogenous factors able to maintain the pluripotent state (the ability of the cells to differentiate into all cell types). For example, addition of leukemia inhibitory factor (LIF) to mouse ES cells can promote the undifferentiated growth of these cells. However, even LIF, the “gold standard” factor for undifferentiated mouse ES cell culture, cannot maintain homogeneous undifferentiated ES cell populations. Neither can it prevent spontaneous differentiation of the cells in suspension culture, a technique essential for obtaining sufficient quantities of pluripotent cells to allow transfer of ES cell therapies into the clinic.
The market readiness of stem cell therapy awaits the development of technologies capable of imparting control and direction on hES cell growth.
Current methods for the derivation and maintenance of stem cells such as embryonic stem cells are technically demanding and inefficient, with a success rate generally less than 30%.
Current laboratory methods used to maintain cultures of proliferative hES cells are also unable to produce such cells fast enough to respond to increasing demand. Current methods rely on recapitulation of the cell-cell and cell-matrix environment and are limited to production in inefficient monolayer culture.
To date pluripotent hES cells have proven difficult to expand in vitro and significant spontaneous differentiation occurs under current “optimal” growth conditions. Leukemia inhibitory factor (LIF) is the current “gold standard” for undifferentiated mouse ES cell culture. However LIF cannot maintain homogeneous ES cell populations, neither can it prevent spontaneous differentiation of the cells.
The growth factor fibroblast growth factor 2 (FGF-2) is also commonly used as a supplement in the culture of human stem cells, such as human embryonic stem cells. This growth factor helps human stem cells to remain undifferentiated and capable of proliferation in culture.
Currently, hES cells are generally grown in direct contact with mouse feeder cells or in media pre-conditioned by nutrient components derived from such cells. Such cells carry the risk of passing microbes and infectious agents to the recipient. As such the FDA has stated that it will demand extensive testing and long term follow up studies on therapies using such technologies. Mechanisms aimed at removing such dangers have thus far focused on the use of expensive growth factor supplements.
To date, none of the prior art techniques have been consistently successful enough to allow their widespread clinical use in biological cell-based therapies. There therefore remains a need to develop improved methods for preparing biological cells for therapeutic use, and improved methods of therapy utilizing biological cells.
Furthermore, it will also be appreciated in the light of the above that there exists a need to develop new or improved cell culture methods, conditions and media capable of promoting biological cell growth without maturation and/or differentiation. Such new or improved cell culture resources may be of use not only in the therapeutic adaptation of stem and/or progenitor cells, but also in the culture of biological cells (and particularly stem and/or progenitor cells) for research and/or development purposes. Although it is desirable to be able to culture such cells (for example to allow expansion of cell numbers without maturation or differentiation) there is a general lack of suitable resources available to the skilled person in the prior art.